Bolstered by the overexpression of Id proteins in a wide range of human tumors and the frequent correlation with a poor prognosis, several investigators have suggested that Id proteins play key roles in the initiation and progression of cancer. Our own work identified the Retinoblastoma tumor suppressor pathway as a network that cross talks with Id proteins during development but is subverted by Id in Myc-driven cancer. However, the post-translational machinery that regulates Id proteins in normal cells is unknown. Similarly, it remains unclear whether genetic alterations leading to deregulated Id activity exist in cancer cells. Recently, we discovered that colon cancer cells acquire a missense mutation in the Id2 gene that converts Threonine-27 into the unphosphorylatable amino acid Alanine. We also found that Threonine-27 is phosphorylated by the Dyrk1 kinases in vitro and in vivo. Our Specific Aims will focus on the regulation and function of this phosphorylation event and the destruction signals that we recently identified in Id proteins. Thus, we will define the signaling pathways converging on Threonine-27 phosphorylation and elucidate how they regulate Id2 protein function. Through biochemical and genetic approaches we will also determine why tumor cells select for the Threonine-27 to Alanine Id2 mutant protein. To address this problem in the context of the whole organism, we will generate knock-in mice that conditionally express the tumor-specific mutation of Id2. Finally, we will take advantage of state-of-the-art mass spectrometry approaches developed in our laboratory to identify and characterize novel enzymes responsible for post-translational modifications of Id proteins. Through the proposed work, we aim to provide a better understanding of the post-translational regulatory machinery for Id proteins and unravel new layers of intervention targeting the alteration of this machinery in human cancer.